pCMV-T7-SpRY-HF1-P2A-EGFP (RTW5008)

LOCUS       pCMV-T7-SpRY-HF1-P2A-EGFP  (RTW5008)                8451 bp ds-DNA     circular SYN 20-MAR-2020
DEFINITION  CMV and T7 promoter expression plasmid for human codon optimized 
            SpRY-HF1(SpRY=A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/
            R1333P/R1335Q/T1337R;HF1=N497A/R661A/Q695A/Q926A) with a c-terminal 
            bi-partite NLS, 3x flag tag, and P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-T7-SpRY-HF1-P2A-EGFP  (RTW5008)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8451)
  AUTHORS   Kleinstiver
  TITLE     Kleinstiver lab CRISPR plasmids
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 8451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 20, 2020 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8451
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        165..544
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        545..748
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     699..719
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     790..809
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        790..808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      816..825
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             822..2540
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTAFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     CDS             4980..5000
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5013..5078
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5097..5153
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5154..5873
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5199..5220)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5460..5479)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5807..5828
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5879..5896
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5919..5936)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5925..6149
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6220..6236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6220..6236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6233..6255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6244..6260
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(6268..6298)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6313..6334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6451..6468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6622..7210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6702..6721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7381..8241)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8004..8023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8242..8346)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8425..8444)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
       61 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
      121 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      181 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      241 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      301 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      361 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      421 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      481 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      541 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      601 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      661 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      721 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc
      781 cgcggccgct aatacgactc actataggga gagccgccac catggacaag aagtacagca
      841 tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg
      901 tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc
      961 tgatcggagc cctgctgttc gacagcggcg aaacagccga gagaacccgg ctgaagagaa
     1021 ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca
     1081 gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg
     1141 tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg
     1201 cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg
     1261 acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc
     1321 acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc
     1381 agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg
     1441 acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg
     1501 cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg
     1561 gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa ctgcagctga
     1621 gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg
     1681 ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga
     1741 gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag agatacgacg
     1801 agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt
     1861 acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag
     1921 ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg
     1981 aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca
     2041 acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg
     2101 aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc
     2161 gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca
     2221 gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac aagggcgctt
     2281 ccgcccagag cttcatcgag cggatgaccg ccttcgataa gaacctgccc aacgagaagg
     2341 tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag
     2401 tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag cagaaaaagg
     2461 ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg
     2521 actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt
     2581 tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc
     2641 tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg
     2701 aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag
     2761 tgatgaagca gctgaagcgg cggagataca ccggctgggg cgccctgagc cggaagctga
     2821 tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg
     2881 gcttcgccaa cagaaacttc atggccctga tccacgacga cagcctgacc tttaaagagg
     2941 acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc
     3001 tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc
     3061 tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga
     3121 accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg
     3181 gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc
     3241 agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg
     3301 aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc
     3361 tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga
     3421 gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc
     3481 tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg
     3541 gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa acccgggcca
     3601 tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg
     3661 acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc
     3721 ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg
     3781 cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg
     3841 agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc
     3901 aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg aactttttca
     3961 agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg
     4021 gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc
     4081 tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca
     4141 aagagtctat cagacccaag aggaacagcg ataagctgat cgccagaaag aaggactggg
     4201 accctaagaa gtacggcggc ttcctgtggc ccaccgtggc ctattctgtg ctggtggtgg
     4261 ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca
     4321 ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct
     4381 acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg
     4441 aaaacggccg gaagagaatg ctggcctctg ccaagcagct gcagaaggga aacgaactgg
     4501 ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg
     4561 gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg
     4621 acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc
     4681 tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg
     4741 agaatatcat ccacctgttt accctgacca gactgggagc ccctagagcc ttcaagtact
     4801 ttgacaccac catcgacccc aagcagtaca gaagcaccaa agaggtgctg gacgccaccc
     4861 tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag
     4921 gcgacggatc cggcggaggc ggaagcggga aaagaaccgc cgacggcagc gaattcgagc
     4981 ccaagaagaa gaggaaagtc tcgagcggag gcgactacaa agaccatgac ggtgattata
     5041 aagatcatga catcgattac aaggatgacg atgacaaggg aggcggtgga agcggcgcaa
     5101 caaacttctc tctgctgaaa caagccggag atgtcgaaga gaatcctgga ccgatggtga
     5161 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     5221 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     5281 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     5341 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     5401 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     5461 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     5521 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     5581 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     5641 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     5701 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     5761 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     5821 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tgaccggtca
     5881 tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     5941 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     6001 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     6061 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     6121 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     6181 gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     6241 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     6301 ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     6361 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     6421 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6481 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6541 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6601 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6661 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6721 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6781 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6841 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6901 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6961 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     7021 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     7081 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     7141 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     7201 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     7261 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     7321 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7381 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7441 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7501 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7561 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7621 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7681 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7741 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7801 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7861 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7921 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7981 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     8041 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     8101 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     8161 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     8221 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     8281 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     8341 tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atcgatctcc
     8401 cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt t
//